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J. Biol. Chem., Vol. 279, Issue 33, 34903-34912, August 13, 2004

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Functional Domains of Brevibacillus thermoruber Lon Protease for Oligomerization and DNA Binding

ROLE OF N-TERMINAL AND SENSOR AND SUBSTRATE DISCRIMINATION DOMAINS^*

Alan Yueh-Luen Lee, Chun-Hua Hsu, and Shih-Hsiung Wu¶

From the Institute of Biological Chemistry, Academia Sinica, Taipei 115 and the Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan

Lon protease is a multifunctional enzyme, and its functions include the degradation of damaged proteins and naturally short lived proteins, ATPase and chaperone-like activities, as well as DNA binding. A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized with an N-terminal domain, a central ATPase domain that includes a sensor and substrate discrimination (SSD) domain, and a C-terminal protease domain. Here we present a detailed structure-function characterization of Bt-Lon, not only dissecting the individual roles of Bt-Lon domains in oligomerization, catalytic activities, chaperone-like activity, and DNA binding activity but also describing the nature of oligomerization. Seven truncated mutants of Bt-Lon were designed, expressed, and purified. Our results show that the N-terminal domain is essential for oligomerization. The truncation of the N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase, and chaperone-like activities but retained the DNA binding activity, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities. We further found that the SSD is involved in DNA binding based on gel mobility shift assays. On the other hand, the oligomerization of Bt-Lon proceeds through a dimer tetramer hexamer assembly model revealed by chemical cross-linking experiments. The results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon, and ionic interactions are mainly responsible for the assembly of hexamers.

Received for publication, March 31, 2004 , and in revised form, June 1, 2004.

^* This work was supported by the National Science Council and Academia Sinica, Taipei, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan. Tel.: 886-2-2785-5696, ext.7101; Fax: 886-2-2653-9142; E-mail: shwu{at}gate.sinica.edu.tw.

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